www.nature.com/scientificreports OPEN Ste2 receptor‑mediated chemotropism of Fusarium graminearum contributes to its pathogenicity against wheat Pooja S. Sridhar1, Daria Trofmova1, Rajagopal Subramaniam2, Dianevys González‑Peña Fundora3, Nora A. Foroud3, John S. Allingham1 & Michele C. Loewen1,4* Fusarium Head Blight of wheat, caused by the flamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. While many studies have addressed the responses of both wheat and F. graminearum during their interaction, the possibility of fungal chemotropic sensing enabling pathogenicity remains unexplored. Based on recent fndings linking the pheromone‑sensing G‑protein‑coupled receptor Ste2 to host‑directed chemotropism in Fusarium oxysporum, we investigated the role of the Ste2 receptor and its downstream signaling pathways in mediating chemotropism of F. graminearum. Interestingly, a chemotropic response of growing hyphae towards catalytically active Triticum aestivum ‘Roblin’ cultivar secreted peroxidases was detected, with deletion of STE2 in F. graminearum leading to loss of the observed response. At the same time, deletion of STE2 signifcantly decreased infection on germinating wheat coleoptiles, highlighting an association between Ste2, chemotropism and infection by F. graminearum. Further characterization revealed that the peroxidase‑directed chemotropism is associated with stimulation of the fungal cell wall integrity mitogen‑activated protein kinase signaling cascade. Altogether, this study demonstrates conservation of Ste2‑mediated chemotropism by Fusarium species, and its important role in mediating pathogenicity. Filamentous fungi grow by extending their hyphal tips to form an extensive mycelial network, with the hyphal tips ofen serving as the frst point of contact with a new environment. Tey respond to changes in their environment by directing hyphal growth towards or away from a range of chemical stimuli. Directed hyphal growth towards a chemical stimulus, known as chemotropism, occurs not only in response to nutrient sources and fungal mat- ing factors secreted from opposite mating type cells, but also towards host organisms that the fungi colonize 1–5. Host-directed chemotropism of fungi is predominantly mediated by G-protein-coupled receptors (GPCRs)5–7, and generally leads to spatial proximity of the fungal cells with the host cells, enabling a direct physical interaction and a complex array of molecular responses that underlie the interaction between the two organisms. GPCRs undergo ligand-mediated conformational changes to transduce extracellular stimuli into intracel- lular signals. Classically, GPCR stimulation by its ligand leads to the dissociation of its associated heterotrimeric G-protein into α and βγ subunits, which then recruit and activate signalling cascades within the cell, ultimately efecting appropriate biological responses 8. Originally, it was widely accepted that GPCRs exist exclusively in either an active or inactive conformation, where one GPCR is activated by one ligand resulting in G-protein- mediated signaling and one distinct biological outcome. However, research on G-protein-dependent versus independent signaling of GPCRs over the past two decades has demonstrated that these receptors can exist in multiple conformations depending on the nature of the bound ligand, with diferent conformations leading to activation of diferent signaling cascades and biological outcomes. Tis phenomenon has been termed ‘biased 1Department of Biomedical and Molecular Sciences, Queen’s University, 18 Stuart St., Kingston, ON K7L 3N6, Canada. 2Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada. 3Agriculture and Agri-Food Canada, 5403, 1st Avenue South, Lethbridge, AB T1J 4B1, Canada. 4National Research Council of Canada, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:10770 | https://doi.org/10.1038/s41598-020-67597-z 1 Vol.:(0123456789) www.nature.com/scientificreports/ GPCR signaling’ (reviewed extensively 9–11). While such research has largely been limited to mammalian systems, biased GPCR signaling has been observed in other phyla with the fungal pheromone receptor, Ste2p, in the model organism Saccharomyces cerevisiae serving as canonical example12. Ste2p, along with the Ste3p receptor, are pheromone sensing GPCRs in S. cerevisiae, encoded by the STE2 and STE3 genes. Tey are expressed on the surface of a- and α-type cells, and recognize α- and a-pheromones secreted by opposite mating type cells, respectively13–15. Te pheromone receptors and peptides expressed in each cell type are dictated by specifc alleles present at the mating-type (MAT) locus, MATα and MATa 16. Although these loci are present in all fungi, the system is best characterized in S. cerevisiae. Classically, binding of α-pheromone peptide to Ste2p activates the pheromone response mitogen-activated protein kinase (MAPK) signalling cascade consisting of Ste11p-Ste7p-Fus1p, leading to cell cycle arrest, shmoo formation and subsequently the formation of a diploid zygote 13. However, multiple studies have suggested the existence of alternate functionalities for S. cerevisiae Ste2p (ScSte2p), specifcally in the mating events that occur downstream of cell cycle arrest, which are infuenced by factors such as pheromone gradients 17–20 as well as localization of the ScSte2p receptor to the mating projection21. Furthermore, specifc mutations in ligand-interacting residues of ScSte2p resulted in dif- ferent efects on G-protein-mediated MAPK signaling and diploid zygote formation12,22,23. In contrast, much less is known about the counterparts of ScSte2p in multicellular fungi where the mating type of an organism is governed by more complex mechanisms, or where mating is not relevant to the fungal life cycle. Within the Fusarium genus, there exists a diversity of homothallic (fungi that can fertilize themselves to undergo sexual reproduction e.g. Fusarium graminearum), heterothallic (fungi that require a compatible partner to undergo sexual reproduction e.g. Fusarium fujikuroi) and even asexual (e.g. Fusarium oxysporum) species, rais- ing the possibility of diverse roles that both pheromones and their receptors may play in fungal biology. Recently, F. oxysporum, a fungal pathogen that causes vascular wilt on many plants including tomatoes24, was found to use the Ste2 (FoSte2) receptor to mediate chemotropism towards the tomato plant roots that it colonizes5. Tis chemotropic growth was shown to be in response to the catalytic product of a tomato root-secreted peroxidase. Rather than activating the pheromone response MAPK signaling pathway, this FoSte2-mediated response was found to be transduced through the cell wall integrity (CWI) pathway, consisting of FoBck1-FoMkk2-FoMpk1, an alternate MAPK signaling pathway in fungi. Furthermore, both FoSte2 and FoSte3 have been shown to regu- late conidial germination through autocrine pheromone signaling in F. oxysporum25. However, whether these alternate functionalities for Ste2 hold true more generally in higher fungi remains to be determined. Fusarium graminearum causes Fusarium Head Blight (FHB) in wheat and other cereal crops, resulting in reduced grain quality and contamination with fungal mycotoxins, leading to severe economic and crop losses worldwide26. While the infection biology of the airborne F. graminearum has been extensively studied and dis- plays notable diferences from that of the soilborne F. oxysporum, knowledge regarding F. graminearum chemo- sensing and any role it may play in initiation of its infection is lacking. In addressing this gap, it is important to note that despite its homothallic nature, Ste2 and its respective phero- mone remain encoded in the F. graminearum genome27 (FgSTE2). Furthermore, F. graminearum also encodes an ortholog to the F. oxysporum CWI signalling pathway MAPK protein (FgMgv1; orthologous to FoMpk1 in F. oxysporum), as well as two other MAPKs, FgGpmk1 and FgHog1. Interestingly, while these MAPKs are pri- marily associated with cell wall integrity and remodelling 28, pathogenicity and invasion 29,30, and osmotic stress response31, respectively, all three have been implicated in pathogenicity (reviewed by di Pietro et al.32). On this basis, wild type and a STE2 deletion mutant of F. graminearum (Fgste2Δ) were comparatively tested for their chemotropic responses to a panel of nutrients, metabolites and peroxidases, as well as for their pathogenicity. Te FgSte2 receptor was found to be essential for sensing a wheat peroxidase-derived chemoattractant and its deletion signifcantly reduced the pathogenicity of F. graminearum on germinating wheat coleoptiles. Observed activities were subsequently linked to phosphorylation of the MAPK FgMgv1, but not FgGpmk1. Together these fndings emphasize the conserved nature of the mechanisms underlying host-mediated chemotropism among Fusarium species. Results Fusarium graminearum exhibits chemotropism towards chemical stimuli. A quantitative chem- otropism assay5 was used to assess the abilities of diferent compounds to induce directional hyphal growth in F. graminearum (Supplemental Figure S1a). Cell concentrations of 0.25 million macroconidia per mL of aque- ous agar media were found to be the most suitable for quantifying hyphae. Higher cell concentrations resulted in intertwining hyphae that could not be counted discreetly, while concentrations lower than 0.25 million per mL yielded insufcient